Studies have been initiated to examine the pathway of secretion of pertussis toxin (PT ) from B. pertussis. Several mutants which lack the ability to secret pertussis toxin have been isolated. These mutations have been mapped to a locus which extends from about 1 kilobases (kb) to 9 kb downstream of the PT structural genes. One of these mutations was characterized in some detail and was found to produce the same amount of PT as the parent strain, yet the PT produced remained cell associated and was not secreted into the medium. Secretion of filamentous hemagglutinin and export of the outer membrane protein pertactin were unaffected in this mutant suggesting that the mutation was specific for PT secretion. The region of the B. pertussis chromosome which appeared to be important for secretion of PT as indicated by mutational analysis was sequenced. Sequence analysis revealed seven open reading frames (ORFs). A search of the Swiss protein data base for proteins homologous to the protein predicted by ORF A did not reveal any good match. In contrast, the proteins predicted by ORF B, C, D, E, F and G were found to be homologous to the VirB4, VirB6, VirB8, VirB9, VirB10, and VirB11 protein, respectively, from the plant pathogen, Agrobacterium tumefaciens. The VirB proteins have been implicated in the transfer of a piece of DNA, T-DNA, across bacterial membranes. After release from the bacterial cell, the T-DNA ultimately crosses plant cell membranes, integrates into the plant cell genome, and codes for biosynthesis of plant growth hormones. Overexpression of these hormones leads to a loss of division control and tumors. Our data suggest that several accessary proteins may be involved in secretion of PT from B. pertussis and that this transport system may be a member of a family of transport systems found in other types of bacteria.